Coumestrol Down-Regulates Melanin Production in Melan-a Murine Melanocytes through Degradation of Tyrosinase.

Pigmentation reflects skin darkening caused by melanin production, but excessive melanin synthesis may cause problems, such as melasma, solar lentigo, dark spots, and freckles. Considerable effort has been devoted to alleviating these undesired symptoms through the development of safe and effective depigmenting agents. Coumestrol, a plant-derived natural isoflavone with an estrogen-like structure and actions, is known to have anti-aging ability, but its potential depigmenting efficacy has not been evaluated. In the present study, we investigated the effects of coumestrol on melanin synthesis in normal melan-a murine melanocytes. Coumestrol significantly reduced melanin synthesis in a concentration-dependent manner up to a concentration of 25 µM without causing cytotoxicity. It also brightened tissue in an artificial skin model (MelanoDerm) that incorporates both human keratinocytes and melanocytes. Interestingly, although coumestrol did not inhibit tyrosinase activity or transcript level in melan-a cells, it clearly decreased the expression level of tyrosinase protein at a concentration of 25 µM. This coumestrol-induced reduction in tyrosinase protein levels was prevented by pretreatment with the proteasome inhibitor MG-132 or the lysosomal proteolysis inhibitor chloroquine. Collectively, our findings indicate that coumestrol exerts an inhibitory effect on melanin synthesis in melan-a cells, at least in part, through degradation of tyrosinase. These findings suggest that coumestrol is a good candidate for use in depigmentary reagents from a cosmetic and clinical perspective.

20-O-ß-d-glucopyranosyl-20(S)-protopanaxadiol, a metabolite of ginseng, inhibits colon cancer growth by targeting TRPC channel-mediated calcium influx.

Abnormal regulation of Ca(2+) mediates tumorigenesis and Ca(2+) channels are reportedly deregulated in cancers, indicating that regulating Ca(2+) signaling in cancer cells is considered as a promising strategy to treat cancer. However, little is known regarding the mechanism by which Ca(2+) affects cancer cell death. Here, we show that 20-O-ß-d-glucopyranosyl-20(S)-protopanaxadiol (20-GPPD), a metabolite of ginseng saponin, causes apoptosis of colon cancer cells through the induction of cytoplasmic Ca(2+). 20-GPPD decreased cell viability, increased annexin V-positive early apoptosis and induced sub-G1 accumulation and nuclear condensation of CT-26 murine colon cancer cells. Although 20-GPPD-induced activation of AMP-activated protein kinase (AMPK) played a key role in the apoptotic death of CT-26 cells, LKB1, a well-known upstream kinase of AMPK, was not involved in this activation. To identify the upstream target of 20-GPPD for activating AMPK, we examined the effect of Ca(2+) on apoptosis of CT-26 cells. A calcium chelator recovered 20-GPPD-induced AMPK phosphorylation and CT-26 cell death. Confocal microscopy showed that 20-GPPD increased Ca(2+) entry into CT-26 cells, whereas a transient receptor potential canonical (TRPC) blocker suppressed Ca(2+) entry. When cells were treated with a TRPC blocker plus an endoplasmic reticulum (ER) calcium blocker, 20-GPPD-induced calcium influx was completely inhibited, suggesting that the ER calcium store, as well as TRPC, was involved. In vivo mouse CT-26 allografts showed that 20-GPPD significantly suppressed tumor growth, volume and weight in a dose-dependent manner. Collectively, 20-GPPD exerts potent anticarcinogenic effects on colon carcinogenesis by increasing Ca(2+) influx, mainly through TRPC channels, and by targeting AMPK.

Identification of sake extract as a new anti-melanogenic ingredient by in vitro and clinical trials.

Overproduction of melanin is the cause of skin hyperpigmentation, which is related to several skin diseases and cosmetic concerns. Sake is a Japanese alcoholic beverage produced from rice and water by fermentation, but is little known for its effect on melanogenesis. To identify the effect of sake extract on melanin synthesis, a melanin assay was performed in melan-A murine melanocytes. Sake extract treatment significantly inhibited melanin production in a dose-dependent manner, and tyrosinase, the rate-limiting enzyme of melanogenesis, decreased significantly at the protein level. Further investigations were performed with multiple assay systems; a sake extract reduced melanin production in melan-A/SP-1 murine cell co-culture, and also in MelanoDerm, a skin equivalent model of human keratinocytes-melanocytes. Finally, subjects were treated with a formula containing the sake extract. Topical application of the sake extract product improved skin lightness (L*) significantly within 7 days. We identified sake extract as a new anti-melanogenic ingredient through in vitro and in vivo experiments. These results suggest that a sake extract can be used to improve skin hyperpigmentation.

Metabolic dependence of green tea on plucking positions revisited: a metabolomic study.

The dependence of global green tea metabolome on plucking positions was investigated through (1)H nuclear magnetic resonance (NMR) analysis coupled with multivariate statistical data set. Pattern recognition methods, such as principal component analysis (PCA) and orthogonal projection on latent structure-discriminant analysis (OPLS-DA), were employed for a finding metabolic discrimination among fresh green tea leaves plucked at different positions from young to old leaves. In addition to clear metabolic discrimination among green tea leaves, elevations in theanine, caffeine, and gallic acid levels but reductions in catechins, such as epicatechin (EC), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epigallocatechin-3-gallate (EGCG), glucose, and sucrose levels were observed, as the green tea plant grows up. On the other hand, the younger the green tea leaf is, the more theanine, caffeine, and gallic acid but the lesser catechins accumlated in the green tea leaf, revealing a reverse assocation between theanine and catechins levels due to incorporaton of theanine into catechins with growing up green tea plant. Moreover, as compared to the tea leaf, the observation of marked high levels of theanine and low levels of catechins in green tea stems exhibited a distinct tea plant metabolism between the tea leaf and the stem. This metabolomic approach highlights taking insight to global metabolic dependence of green tea leaf on plucking position, thereby providing distinct information on green tea production with specific tea quality.

Geographical and climatic dependencies of green tea (Camellia sinensis) metabolites: a (1)H NMR-based metabolomics study.

The effects of climatic conditions on green tea metabolites in three different growing areas of Jeju Island, South Korea, were investigated through global metabolite profiling by (1)H nuclear magnetic resonance (NMR) spectroscopy. Pattern recognition methods, such as principal component analysis (PCA) and orthogonal projection on latent structure-discriminant analysis (OPLS-DA), revealed clear discriminations of green teas from the three different growing areas. Variations of theanine, isoleucine, leucine, valine, alanine, threonine, glutamine, quinic acid, glucose, epicatechin (EC), epigallocatechin (EGC), epigallocatechin-3-gallate (EGCG), and caffeine levels were responsible for the discriminations. Green teas grown in an area with high temperature, long sun exposure time, and high rainfall had higher levels of theanine but lower levels of isoleucine, leucine, valine, alanine, EC, EGC, EGCG, and caffeine than those grown in areas with relatively low temperature, short sun exposure time, and low rainfall. These results indicate that high temperature, long sun exposure, and high preciptation stimulate theanine synthesis in green tea during the spring season. This study highlights how metabolomics coupled with multivariate statistical analysis can illuminate the metabolic characteristics of green tea associated with climatic variables, thereby allowing for the assessment of quality strategy in green tea production.